JPS58141798A - Analysis of polyamine - Google Patents

Abstract

PURPOSE:To determine a polyamine, in high reproducibility, by using a putrescine.oxidase derived from Micrococcus flavidus as an enzyme and trichloroacetic acid as a desorbing agent. CONSTITUTION:A polyamine such as spermidine, putrescine, etc. is adsorbed to an adsorbent, preferably a carboxylic acid-type weakly acidic anion exchange resin, and desorbed by using trichloroacetic acid as the desorbing resin, and desorbed by using trichloroacetic acid as the desorbing agent. The resultant polyamine solution is made to react with a putrescine oxidase derived from Micrococcus flavidus preferably at 7.0-8.5pH and 10-40 deg.C. The produced hydrogen peroxide is determined by colorimetry using a proper reagent.

Description

[Detailed Description of the Invention] This invention relates to a method for analyzing ions. Although dllJ amine is widely distributed in the living world, it is viewed medically as a nuisance because its content is high in rapidly proliferating cells, especially in tumor cells. Additionally, the content of 4-lyamine in body fluids such as blood and urine of cancer patients is higher than that of healthy people.
, was shown by Russell et al. Since then, many researchers have investigated the correlation between cancer and the concentration of polyamines in body fluids, and the validity of the research results of Russell et al. has been confirmed.

However, it is difficult to quantify polyamines in body fluids.
This is extremely difficult because it has a low carbon content and contains various impurities other than 4 rea.

Currently, the most reliable polyamine quantitative method uses high performance liquid chromatography. However, since the time required for analysis using this method is more than one hour for one sample, it is still beyond the scope of an analytical method that can be used up to the research level. Enzymatic methods are attracting attention for this purpose.
Principle Fi of polyamine quantitative analysis using an enzymatic method: A polyamine-degrading enzyme is used to obtain a product that is easier to detect than a polyamine, and the product is then detected.

However, the problem is which polyamine-degrading enzyme to select as the polyamine-degrading enzyme in quantifying 1-lyamine using an enzymatic method. One example is gutreon oxidase. The element Gtolemine oxidase was discovered in Micrococcus Φo-zeus by Fu Adachi [Agricultural Research Journal, Vol. 30, /2θco(/96&)]. However, when the enzyme obtained from Micrococcus fistulae is applied to a polyamine footrest by an enzymatic method, it has the following drawbacks.

t), (11 Micrococcus roseus has a large amount of putrescine oxidase that accumulates in the bacterial body, and the preparation of the # element is complicated. (2) The cells of Micrococcus roseus are very strong. , - When extracting putrescine oxidase from the body, special equipment must be used to crush the noodle bodies and extract the enzyme. (3) Michaelis constant for putrescine of putrescine oxidase obtained from Micrococcus roseus Because the value is relatively large (/, 2
When quantifying M), the disadvantage is that the enzyme reaction rate is slow and therefore the quantification time is long.

In order to solve the above three drawbacks, namely, the low yield of putrescine oxidase, the difficulty in crushing it itself, and the large m value of the Michaelis constant for putrescine, we developed various microorganisms. Testing for putrescine oxidase in the body 1. Enzyme productivity is more than 30 times higher than that of Micrococcus roseus, the enzyme specific activity in the im body is more than 3 times higher, and furthermore, enzyme purification is easy due to easy cell disruption, and enzyme recovery is easy. He invented and previously proposed a method for producing a large amount of citrescine oxidase (Japanese Patent Application No. 1989-92792, Japanese Patent Application Laid-open No. 1983-9279).

The present invention is a method for analyzing polyamines using Micrococcus frapimes putrescine oxidase obtained by the above production method.

In general, quantitative analysis of polyamines using an enzymatic method involves, for example, adsorbing polyamines in urine to a suitable adsorbent, desorbing the polyamines using a desorbent, and then treating the resulting polyamine solution with peroxidation. It is carried out by colorimetric determination of hydrogen. However, when micrococcus 7rabidas putrescine oxidase is used as the enzyme, and when an acid solution such as hydrochloric acid or sulfuric acid, which is conventionally used as a desorbent, or an aqueous solution containing salts such as common salt is used as a desorbent, spermidine is removed. It has been found that, depending on the type of polyamine VC, there are drawbacks such as unstable color intensity in colorimetric determination and lack of dust repellency.

The present invention has been made to solve this problem of t+Ua.

That is, the present invention is an analysis of polyamines, which is performed by desorbing polyamines from an adsorbent containing polyamines with an iron layer using a desorbent, and detecting 1 g of peroxide water produced by fully acting on the resulting polyamine solution with an enzyme. This is a polyamine analysis method characterized by using trichloroacetic acid ill as a desorbent and using Micrococcus flavimes putrescine oxidase as an enzyme.

Examples of polyamines include spermidine, putrescine, spermine, etc., but spermidine is particularly effective for use in the present invention, and spermidine will be described below as a representative polyamine.

Spermidine For example, when quantifying hydrogen peroxide produced by the action of an enzyme on spermidine in urine, the urine containing spermidine is brought into contact with a suitable adsorbent in order to remove substances that interfere with color development. Adsorb spermidine,
After separating and removing the substances that interfere with color development, a desorption agent is used to obtain a solution containing no substances that interfere with color development.

Adsorbents are not particularly limited, and include cation exchange resins,
Examples include cellulose ion exchangers and others, but preferably cation exchange resins are used. Various ordinary cation exchange resins are used as the cation exchange resin, but Cal-1 is used because it can desorb spermidine under relatively mild conditions. A weakly acidic cation exchange tree of rRW is particularly preferably used. In addition, as a urine sample, unreleased urine as collected may be used, but for example, acyl I lyamine fist Y H V lorase (Japanese Patent Application Laid-open No. 5B
Urine that has been hydrolyzed using -ilIdaogg> may also be used.

The greatest feature of the present invention is that Micrococcus-7 is used as an enzyme.
In addition to using Ravidus-based putrescine oxidase, trichloroacetic acid is used as a desorption agent. Trichloroacetic acid can be arbitrarily used, such as a commercially available product or a special grade for biochemistry, and its concentration is usually O-7 to ON, but especially 0.2 to 0.6N.
Those are preferably used. The amount of trichloroacetic acid used usually ranges from 2 to 30 volumes per volume of adsorbent.

In addition, the desorption method using trichloroacetic acid is the same as the conventional method using hydrochloric acid, sulfuric acid, etc. If the adsorbent is packed in a column etc., the desorbent t
It is best to supply it from the top of the column. Micrococcus flavidus putrescine oxidase is applied to spermidine obtained by desorption using trichloroacetic acid.

Micrococcus 7rabidas putrescine oxidase is a putrescine oxidase obtained from a culture of Micrococcus flavidus.The outline of the production method will be described later, but the details are as described above by the present inventors. etc. suggested it first, and I\j Kaisho! ;7-/Z9Zada is Kinchi. Miku 0:r7kas@7rabiday
, (Mlcrococcus Flavl-dus
) Fi, as described in the above-mentioned Japanese Patent Application Laid-Open Publication No. Sho sq-itrqg II, it is recognized as a new species belonging to the genus Micrococcus discovered by the present inventors, and it has been newly named and the microorganism storage entrustment application acceptance number No. 5633, which has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology.

When treating a spermidine solution obtained by desorption using trichloroacetic acid with the enzyme cytoresine oxidase of Krococcus 7rabidas, it is usually necessary to add a spermidine bath solution using an alkaline solution such as a tris (human tetraxymethyl) amitumene solution. After neutralization, the enzyme is allowed to act. Generally, the ai1 degree of the tris(human tetraxymethyl)anthumethane solution is preferably used at a molar concentration of 0.2 to 0.2 to 0.0 molar.

In addition, the reaction conditions when the spermidine solution is made to act with Micrococcus 7rabidas putrescine oxidase are not particularly limited, but 1 reaction solution OpH cuff, 0
-ff, j Especially '7. j to t and O are preferable because the reaction rate is fast. Other conditions are fI! It is preferable to carry out the process at a temperature of 70 to 0°C, especially to satisfy the conditions of -0 to 3k''C. When oxidase is activated, hydrogen peroxide is produced by reducing the amount of spermidine contained in the spermidine bath solution and reducing the amount of spermidine contained in urine. Colorimetric determination may be performed using a reagent.As the reagent, a quaaminoantipyrine/dichlorophenol/peroxidase type reagent is usually preferably used.

The most effective feature of the present invention is that color intensity is stabilized and reproducibility is achieved in colorimetric determination.

That is, as mentioned above, putrescine oxidase from the Electrocrococcus 7rabidas family is advantageous in terms of enzyme productivity, enzyme specific activity, and cell disruption; When using liquids such as hydrochloric acid or sulfuric acid, or aqueous solutions of salts such as common salt, the color intensity is unstable and is not reproducible. In practice,
It's impossible. However, when trichloroacetic acid is used as a desorbent, a color intensity proportional to the amount of spermidine contained in urine is always obtained. Such a unique phenomenon when using citresine oxidase of the Krokotsucus 7rabidas family is completely unexpected. The reason why such a unique phenomenon occurs is not clear, but the inventors of the present invention speculate as follows. In other words, under normal conditions, putrescine oxidase of the Micrococcoccus heptarabimes system not only oxidizes the co-anno group of subermidine, but also the terminal 7-ary amino group, but in the presence of trichloroacetic acid, it oxidizes spermidine. It is estimated that only the co-class anno group is selectively oxidized and 1 equivalent of hydrogen peroxide is stably generated per 1 equivalent of spermidine.

In addition, Micrococcus 7. lapidus used in the present invention -
Scientific property 3I is as shown below. In addition, the color display is based on "Color Standard J (published by E1 Honshokusha, 1qsi edition)"
I followed.

mouth i form Meat juice and meat juice agar medium were used as media.

■ Cell shape and size spherical. From the early to middle stage of culture, it becomes double balls. O,S
~θ, 9 prn (diameter) Q) Cell pleomorphism
′ None ■ No spores (no flagella) q) No spores ■ Durham staining positive ■ Acid-fast staining negative (2) Growth status in each medium ■ Good growth in broth agar plate culture, round, convex, opaque, dark brown (dull y
yellow), no production of soluble pigment.

0 Meat juice agar slant culture. Good growth, linear growth, opaque, dull yellow.
yellow), no formation of soluble pigment.

■ Meat juice liquid culture grows well, is uniformly cloudy, does not form a fungal film, does not form sediment, and does not form segments.

■ Meat juice gela □ Chin puncture culture Growth on the surface, moderate growth, no liquefaction of gelatin.

■ Ridmus milk culture Alkaline, no bulk liquefaction or coagulation.

13+ Physiological properties■ Nitrate reduction positive■ Denitrification reaction negative) MR test negative) VP test negativeCy Indole formation
Negative 9 Production of hydrogen sulfide Negative ■ Hydrolysis of starch Negative ■ Utilization of citric acid Positive ■ Utilization of inorganic base source Positive [phase] Production of pigment Negative Production of O urease Positive O oxidase Negative 0 Catalase Positive 0 Growth range pH ! ;, 0-10.0 Temperature Ko~3g℃ @ Duty to oxygen Aerobic @ O-F test (Hugh Lelfson method) Does not decompose sugars 0 Production of acid from sugars Gas generation Fi subject to all sugars Also, putrescine and
Oxidase can be obtained as follows.

First, the above-mentioned microorganism is cultured in a medium containing putrescine using a conventional method for producing enzymes and the like.

Liquid aeration culture is advantageous as the form of culture. As the nutrient source for the medium, those commonly used for culturing microorganisms are widely used. The carbon source may be any assimilable expanded hydrogen, such as glucose, molasses, glycerin, etc. As the nitrogen source, any available nitrogen compound may be used, such as ibutone, yeast extract, meat extract, corn, staplei, liquor, ammonium sulfate, ammonium chloride, and the like.

In addition, salts such as common salt, potassium chloride, magnesium sulfate, potassium phosphorus, and dipotassium phosphate are used depending on the membership fee.

In addition, by adding putrescine or spermidine to the medium, the production of putrescine oxidase during culture can be suppressed.
It is preferable to increase the performance. When putrescine and spermidine to be added are compared, 1-trescine is more advantageous in terms of price and the effect of increasing the production capacity of 1-trescine oxidase. The amount of putrescine or spermidine to be added is in the range of O,OS to /.011, preferably about 1 to θ,/ to θ,j%. The culture and temperature may be within a range that allows growth of 1 and production of putrescine oxidase, but preferably a temperature of 35°C to 35°C. The culture time varies depending on the feeding conditions, but it is usually about S to 30 hours, and the growth of the bacteria is in the stationary phase (5tatlonary phase).
), putrescine oxidase production will reach its maximum if the cultivation is terminated at the next stage. In the thus obtained culture, putrescine oxidase is contained and accumulated within the bacterial cells.

In order to extract putrescine oxidase from the culture thus obtained and obtain a crude putrescine oxidase-containing solution, for example, the culture is first subjected to solid-liquid separation by means of centrifugation. The obtained wet bacterial cells are suspended in a phosphate buffer solution, Tris-hydrochloric acid buffer, etc., depending on the membership fee.
Next, p-putrescine oxidase is extracted from the whole by appropriately selecting and combining integral treatment means such as ultrasonic crushing treatment, crushing treatment with Dynomill, and lysozyme treatment, to obtain a crude putrescine oxidase-containing liquid.

Further, purified putrescine oxidase can be obtained by treating this crude putrescine oxidase-containing solution using known methods for isolating proteins, enzymes, etc.

Next, Examples and Comparative Examples will be given, but the present invention is not limited thereto.

Example-7 Urine lOθJ was collected and diluted with /M Tris-HCl buffer (pHf
, O) to adjust the pH to 70g, then J*000
To the supernatant from which the precipitate was removed by centrifugation at rpm for 10 minutes, an aqueous spermidine solution was added to a final concentration of OsM.

1 ml of the thus prepared urine sample solution was passed through a mini-column (resin amount: 0.2 u) of a cation exchange resin Piorec 70 (2000) (manufactured by Io-Rad Co., Ltd., Carunic Acid W).

After washing the mini column with ion-exchanged water S, θV, 0.2N
Elute with trichloroacetic acid, eluent 3. Obtained OV.

Spermidine in the eluate was quantified enzymatically using putrescine oxidase purified from Micrococcus flavidus. Detection is done by pretosine oxidase (
This was carried out by coloring the hydrogen peroxide produced by the action of lumidine using a deraminoantiphosphorus/dichlorophenol/beroxidase system.

Assay solution 0.1M Tris-HCl buffer 70 fusion guaminoantipyrine dichlorophenol
90g of peroxidase and 114 volumes of pretocin oxidase 100unl
t s eluate/, 0atliCθ, jM) Lith solution o
, after neutralization by adding isV, the above assay solution/, 0*
t was added to react at 3θ°C, and the absorbance of j/Q nm was measured at 20D514. As a result, as shown in Table 1, a constant absorbance was exhibited approximately 30 minutes after the start of the reaction, and the value remained stable without faeces even after 60 minutes.

Furthermore, Fig. 1 shows how the absorbance changes over time.

Comparative Example-7 Among the compounds shown in Example-1, o, :t'x trichloroacetic acid was added to 0. The same operation as shown in the example was carried out except that N-hydrochloric acid was used. Absorbance at r/4' nm: 005
As shown in Table 2, 14 did not show a constant value and the absorbance continued to increase even 6θ minutes after the start of the reaction.

In addition, Fig. 1 shows how the absorbance changes over time.

Example - One Example - The final suilumidine concentration was O%l.
30
Minutes later! The absorbance at r/II nm; 0D514 was measured and the results are shown in Table 3.

゛Surmidine concentration: C (mM) and oO5 with absorbance 0.022 in case of spermidine concentration O as a blank value
The result of finding the relational expression with 14 was as follows.

Example 3 The same operation as shown in Example 7 was performed except that the concentration of sulmidine added was changed to an unknown concentration, and the absorbance of silInm after 30 minutes was measured. : OD5,4 was determined, and urinary spermidine was quantified using the calibration 1st of formula (1) determined in Example-C.

At the same time, the spermidine solution desorbed by trichloroacetic acid was subjected to high performance liquid chromatography (HPLC).
) The urinary spermidine concentration was determined by analysis.

The results of both analyzes are shown in Table DA.

@da table

[Brief explanation of the drawing]

Figure 1 shows 0.2N trichloroacetic acid and 0.2N trichloroacetic acid as a desorbent.
．． This book compares the color stability when using 2N hydrochloric acid. Figure 1 0 70 20 30 45 6081 Ougin r1 (Written amendment of the division procedure dated June 1980, Mr. Kazuo Wakasugi, Commissioner of the Japan Patent Office 1, Indication of the case, Patent Application No. 22699 of 1981 2, Name of the invention Polyamine Analysis method 3. Person making the amendment, case and relationship Patent applicant address 1-1 Mikage-cho, Tokuyama City, Yamaguchi Prefecture Tokuyama Soda Co., Ltd. Tokyo Headquarters Patent Information Department Telephone number 591-9361 4. Date of amendment order Voluntary, 1 '□b, [Detailed Description of the Invention] and ``Brief Description of Drawings 1'' in the Statement of Subject of Amendment, I17F 2.16, Contents of Amendment (1) Page 2, Line 3 of the Specification ``t9'rs year'' is changed to ``19''
'71' K correction. (2) “Ptolemin” on the 3rd and 4th lines of the same page 3
is corrected with “putrescine” K. (3) Correct "Iological" on page 3, line 7 of the same page to "Nokiological". (4) 4th line: Correct "Yokoashi" in the 9th line to "Search". (5) The "No." on page 4, line 15 is amended to "No. 18984." (6) 11 on page 7, line 12. Although it is ON,
In particular, 0.2 to 0.5 NJ is 1” 1.0 M,
In particular, it is corrected to 0.2 to 0.5 NJ. (7) "Putrescine" on page 10, line 15 is corrected to "putrescine". (8) Page 15, lines 4-5, “Kone, Stapi,
Correct "Corn Steep Liquor" to "Corn Steep Liquor". (9) "Processed lysozyme" on page 16, line 12 of the same page is corrected to "processed, lysozyme." 01 Correct "Nokuiorex 70" on page 17, line 8 of the same page to "Biorex 70". αυ IO, 2NJ on page 17, line 12 of the same page is “0.2M
JK correction. a. Correct "pretoscine" on page 17, line 16 of the same page to "putrescine." αj ``0.2NJ is l'-0,2'' on page 20, line 4 of the same page.
Correct to "M". I, page 24, line 2 [m M J is corrected by rμMJK. αji Same page 25, line 10, 10.2NJ is “0.2
Correct to "M". that's all

Claims (1)

[Scope of Claims] ... - Polyamines are desorbed from an adsorbent on which lyamines have been attached in a desorbent, and hydrogen peroxide produced by reacting an enzyme to the resulting 4lyamines solution is detected. An analytical method for amines characterized by using trichloroacetic acid as a desorbent and Micrococcus flavidus putrescine and oxidase as enzymes. 1. The analytical method according to claim 01, wherein the polyamine is spermidine. (31a) The analysis method according to claim 11, wherein the adherent is a cation exchange resin.